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Title: N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803

Abstract

Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide withmore » the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less

Authors:
 [1];  [1];  [1];  [1];  [2]
  1. Louisiana State Univ., Baton Rouge, LA (United States)
  2. Univ. of California, Davis, CA (United States)
Publication Date:
Research Org.:
Louisiana State Univ., Baton Rouge, LA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1337766
Grant/Contract Number:
FG02-98ER20310; FG02-09ER20310; FG02-09ER16070; SC0002628
Resource Type:
Journal Article: Published Article
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 11; Journal Issue: 9; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; lipids; signal peptides; peripheral membrane proteins; synechocystis; signal processing; proteases; membrane proteins; outer membrane proteins; 60 APPLIED LIFE SCIENCES; Peripheral Membranes; Synchocystis

Citation Formats

Juneau, Andrea D., Frankel, Laurie K., Bricker, Terry M., Roose, Johnna L., and Theg, Steven M.. N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0163646.
Juneau, Andrea D., Frankel, Laurie K., Bricker, Terry M., Roose, Johnna L., & Theg, Steven M.. N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803. United States. doi:10.1371/journal.pone.0163646.
Juneau, Andrea D., Frankel, Laurie K., Bricker, Terry M., Roose, Johnna L., and Theg, Steven M.. 2016. "N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803". United States. doi:10.1371/journal.pone.0163646.
@article{osti_1337766,
title = {N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803},
author = {Juneau, Andrea D. and Frankel, Laurie K. and Bricker, Terry M. and Roose, Johnna L. and Theg, Steven M.},
abstractNote = {Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.},
doi = {10.1371/journal.pone.0163646},
journal = {PLoS ONE},
number = 9,
volume = 11,
place = {United States},
year = 2016,
month = 9
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1371/journal.pone.0163646

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  • Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 tomore » eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less
  • The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminatemore » the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less
  • In higher plants, algae, and a wide variety of prokaryotes, the tetrapyrrole pigment precursor {delta}-aminolevulinic acid (ALA) is formed from glutamate by a process requiring at least three enzymatic components plus tRNA{sup Glu}, ATP, NADPH, M{sup 2+}, and pyridoxal phosphate. Previous reports have shown that the cyanobacterium Synechocystis sp. PCC 6803 produces ALA via the glutamate pathway. We report here the separation of this activity into three enzymatic fractions. Cells were harvested and broken in a French pressure cell. Protein precipitating between 30-55% (NH{sub 4}){sub 2}SO{sub 4} saturation were separated on Blue-Sepharose into unbound protein (fraction I) and bound proteinmore » which was further separated on 2{prime}-5{prime} ADP-agarose into unbound protein (fraction II) and bound protein (fraction III). Reconstitution of ALA-forming activity protein (fraction II) and bound (fraction III). Reconstitution of ALA-forming activity required all three fractions. Only fraction II had the ability to aminoacylate tRNA with glutamate in the presence of ATP and Mg{sup 2+} and therefore contains the glutamyl-tRNA synthetase. After reisolation by phenol extraction, the glutamyl-tRNA could be used as substrate for ALA formation in incubations containing fractions I and III. Only fraction I had the ability to convert synthetic glutamate-1-semialdehyde into ALA. This enzyme activity was 90% inhibited by 5{mu}M gabaculine. The mol wts of the activities in fraction I and II were estimated by Sephadex G-150 gel filtration to be 99,000 and 63,000 (+ 5,000), respectively. The results indicate that the protein components of the ALA-forming pathway in Synechocystis are similar to those of higher plants and eukaryotic algae.« less
  • Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 x 10/sup 8/ cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmidmore » is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV- inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 x 10/sup -4/ and 1.5 x 10/sup -5/, respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.« less